In stem cell research, studies have been done to study the regenerative properties of human mesenchymal stem cells (MSCs) from bone marrow for the cells' potential therapeutic applications. MSCs are a promising source of adult stem cells for regenerative medicine, however many senescent cells are found in the heterogeneous ensemble of progenitors and lineage-committed cells that are associated with loss of proliferation potential and differentiation potential. Regenerative properties are highly variable among MSC subsets. Consequently, identification and isolation of progenitor subsets in heterogeneous MSC cultures are essential to the development of highly efficacious stem cell therapies. In other words, the elimination of senescent cells from heterogeneous MSC cultures may improve the treatment outcome of autologous MSC therapies by increasing both cell yield and enhancing the integrity of regenerated tissue.
Every human cell has cellular surface markers and receptors which identify it to other cells in the body. In 1999, Pittenger et al. were the first to identify antigens that could be used to reproducibly identify MSCs with similar properties. MSC population homogenization is achieved by detecting surface antigens using fluorescently labeled antibodies and fluorescence-activated cell sorting (FACS). These advancements, as stated above, may remove the negative effects of senescent cells in MSC populations and increase the efficacy of regenerative medicine by enriching healthy cell population.
To separate the senescent cells from heterogeneous MSCs from bone marrow, bio-markers such as antigens Neuron-Glial Antigen 2 (NG2) and Melanoma Cell Adhesion Molecule (CD146) have been discovered. Flow cytometry is used in the sorting process. Cells with high surface expression of the antigen and cells with low surface expression of the antigen can be differentiated and separated by the flow cytometer. This method selects a proliferative phenotype from heterogeneous MSCs during ex vivo expansion. Russell et al., Cell-Surface Expression of Neuron-Glial Antigen 2 (NG2) and Melanoma Cell Adhesion Molecule (CD 146) in Heterogeneous Cultures of Marrow-Derived Mesenchymal Stem Cells. Tissue Engineering: Part A, Vol. 19, No. 19-20, 2013.
Another way to characterize cellular senescence is by using a proliferation dye. This dye can be used to measure the doubling time for MSCs of different sizes and granulation. A parent population is first dyed and then allowed to grow. As the cells divide, the dye is distributed approximately evenly amongst the daughter cells. Cells with a lower doubling time will therefore contain less dye after a given amount of time than cells with a high doubling time. This is because cells that divide rapidly will have distributed the dye amongst more daughters. If a fluorescent proliferation dye is chosen, FACS can be employed to separate subpopulations that either divide rapidly or slowly. A schematic representation of how proliferation dye works to identify rapidly and slowly dividing cells is presented in FIG. 1.
Russell et al. found that NG2 expression is uniquely correlated to rapidly dividing stem cells. Sorting using this surface receptor will enrich the sorted population by concentrating the number of rapidly dividing MSCs. Alternatively, sorting by using the death receptor can result in an enriched parent population using a totally different marker. If the death receptor indicates slowly dividing or senescent cells, then removing these cells from the population will enable the parent population to be more robust. As mentioned above, a boosted population is extremely desirable for stem cell therapies.
A current barrier to realizing the therapeutic potential of MSCs is the inability to identify different MSC populations in a heterogeneous culture. The heterogeneous cultures which include cells with lower proliferation and multipotent potential results in substantial variation and decreases the effectiveness of stem cell therapies with MSCs. Previously, this obstacle was addressed by previously unknown identification of two biomarkers, NG2 and CD146, which select for highly proliferative multipotent MSCs. However, identifying and isolation of early aging MSCs is even more desirable to obtain MSCs with higher regenerative potential and better therapeutic efficacy.
In 2010, Wagner et al states that “No date no specific molecular marker is available that prospectively reflects the degree of cellular aging in MSC.” Wagner et al., How to Track Cellular Aging of Mesenchymal Stromal Cells? Aging, April 2010, Vol. 2, No. 4, 224-230.
Secchiero et al. in 2008 found that MSCs express CD264, but did not identify any specific function or properties thereof. Secchiero et al, Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Promotes Migration of Human Bone Marrow Multipotent Stromal Cells. Stem Cells 2008; 2955-2964 (“the expression of surface TRAIL-R2 and TRAIL-R4 was a general feature of MSCs,” where TRAIL-R4 is an acronym of CD264).
Ren et al. in 2011 found that CD264 is expressed at late passage (passage 5-11) MSCs, but not expressed in passage 3. Ren et al., SENESCENCE OF CULTURED BONE MARROW STROMAL CELLS. Biology of Blood and Marrow Transplantation, Feb. 2011; 17(2) (“Flow cytometry analysis confirmed greater expression of TNFRS 1 OD on late passage cells,” where TNFRS 10 D is an acronym of CD264).
Further, it has been suggested that CD264 is widely expressed in tissues and therefore is not senescence-related. Degli-Esposti et al., The Novel Receptor TRAIL-R4 Induces NF-KB and Protects against TRAIL-Mediated Apoptosis, yet Retains an Incomplete Death Domain, IMMUNITY (1997), Vol. 7, 813-820. It was also suggested that CD264 is upregulated by oncogene-induced senescence, but “none was found upregulated in association to replicative senescence.” Collado et al., Senescence in premalignant tumours, Nature (2005), Vol. 436, p. 642 (Supplementary Information).
Zhu et al. further suggested that CD264 is upregulated by stress-induced premature senescence. Zhu et al., Effects of estrogen on stress-induced premature senescence of vascular smooth muscle cells: A novel mechanism for the “time window theory” of menopausal hormone therapy, Atherosclerosis 215 (2011) 294-300.
Lastly, Kim et al. reported that CD264 expression may “depend upon the specific cellular context and may not be a general marker for cellular senescence.” Kim et al., Evaluation of premature senescence and senescence biomarkers in carcinoma cells and xenograft mice exposed to single or fractionated irradiation. Oncology Reports 31: 2229-2235, 2014. This reference teaches away from using CD264 as a marker for early aging MSCs.